Molecular Cancer Research 3:42-49 (2005)
© 2005 American Association for Cancer Research
Signaling and Regulation
Inhibition of I
B
Nuclear Export as an Approach to Abrogate Nuclear Factor-B–Dependent Cancer Cell Survival 1
Shelby O'Connor,
Stuart Shumway and
Shigeki Miyamoto
Program in Cellular and Molecular Biology, Department of Pharmacology, University of Wisconsin-Madison, Madison, Wisconsin
Requests for reprints: Shigeki Miyamoto, Program in Cellular and Molecular Biology, Department of Pharmacology, University of Wisconsin-Madison, 3795 Medical Sciences Center, 1300 University Avenue, Madison, WI 53706. Phone: 608-262-9281; Fax: 608-262-1257. E-mail: smiyamot{at}wisc.edu
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Abstract
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Deregulation of the transcription factor nuclear factor-
B (NF-B) leading to its constitutive activation is frequently observed in human cancer. Because altered NF-B activities often promote the survival of malignant cells, its inhibition is regarded as a promising anticancer strategy. Because activation of the latent cytoplasmic NF-B complex can be induced by a wide variety of different stimuli, its deregulation may occur by an equally large number of distinct mechanisms. This diversity raises a conundrum in conceptualizing general approaches to attenuate NF-B activity in cancer. Here, we provide evidence that inhibition of IB
nuclear export is a viable target to generally abrogate constitutive NF-B activity in different cancer cell types. We show that inhibition of IB nuclear export has an important course of events in cancer cells harboring constitutive NF-B activity—an initial increase in the pool of stable nuclear NF-B/IB complexes that leads to a reduction of constitutive NF-B activity and subsequent induction of apoptosis. Importantly, similar effects on multiple different cancer cell types indicate that inhibition of nuclear export of IB leads to broad inhibition of constitutive NF-B activation regardless of various deregulated, upstream events involved.
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Introduction
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Mammalian cells contain five members of the nuclear factor-B (NF-B) family, RelA (p65), c-Rel, RelB, p50 (NF-B1), and p52 (NF-B2), which form homodimeric or heterodimeric transcription factors (1). NF-B activity is regulated by its association with members of the IB family of inhibitor proteins, such as IB and IBß. An array of extracellular stimuli causes the release of active NF-B via diverse signaling cascades leading to the activation of the cytoplasmic IB kinase (IKK) complex. IKK-dependent phosphorylation of the IB proteins then leads to their ubiquitylation and proteasome-dependent degradation that releases free NF-B. Free NF-B then migrates into the nucleus and activates transcription of genes involved in inflammation, apoptosis, and immune cell development (1).
Although NF-B activity is normally tightly controlled, alterations of the NF-B signaling pathway frequently lead to its constitutive activation in cancer cells (1). Because NF-B can foster antiapoptotic conditions through antiapoptotic gene transcription, it is frequently observed that constitutive NF-B activity promotes cancer cell survival. For example, oncogenic Ras and the Bcr-Abl oncoprotein are found to promote cancer cell survival through constitutive NF-B activation (2, 3). Additionally, primary multiple myeloma cells maintain NF-B activity by constitutive IKK activation (4). Constitutive NF-B activity is also documented in breast, prostate, and head and neck lymphomas and other forms of human malignancies via the chronic activation of Akt, IKK, or other unknown mechanisms (1, 5-8). Production of autocrine factors by certain cancer cells is also linked to constitutive NF-B activation (1). Thus, although NF-B inhibition may be an attractive target for drug development to induce cancer cell death, the diverse mechanisms that cause its activation make it difficult to develop a general strategy. Currently, the inhibitors of the IKK and the proteasome, two of the common cellular factors involved in NF-B signaling events, have been successfully developed (1, 9).
One integral component of normal NF-B regulation is the autoregulatory negative feedback inhibition of continuous activity through NF-B-directed synthesis of IB (10). Newly synthesized IB enters the nucleus, removes NF-B from the DNA, and then exports it out to the cytoplasm to restore the pool of inactive NF-B/IB complexes for subsequent activation (11). Export of IB is primarily mediated by a leucine-rich NH2-terminal nuclear export sequence (N-NES). The IB N-NES is also important for the export of inactive NF-B/IB complexes that move into the nucleus (12-14). With the use of leptomycin B (LMB), a bacterial metabolite with antifungal and antitumor activities (15), it was found that nuclear export of IB is mediated through a CRM-1-dependent pathway (13). Because negative feedback regulation of NF-B activation by IB seems intrinsic to this system in different cell types, various mechanisms are described in cancer cells to counteract this process and maintain chronic NF-B activity. These mechanisms include the activation of upstream signaling events to continually degrade IB via the IKK proteasome (1) or the proteasome inhibitor resistant pathway (16). Mutations in IB (17) can also prevent NF-B inhibition, and hypophosphorylation of IBß, a nonshuttling IB family member, has been described to sequester NF-B in the nucleus away from IB-mediated inhibition (18). In addition, other currently unknown mechanisms may counter this negative feedback regulation.
Previous studies have shown that nuclear trapping of NF-B/IB complexes through mutation of the IB N-NES or treatment with LMB can reduce inducible NF-B activation in response to various stimuli (13). We wanted to determine whether inhibition of IB nuclear export could be used as a strategy to reduce constitutive NF-B activity and induce apoptosis in cancer cells. Our studies in multiple cancer cell lines support the notion that nuclear trapping of IB generally inhibits constitutive NF-B activity and promotes apoptosis. The salient feature of this strategy is that these effects are manifested regardless of various upstream mechanisms involved in the maintenance of constitutive NF-B activation.
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Results and Discussion
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Maintenance of Constitutive NF-B Activity Requires Nuclear Export of IB
To determine whether nuclear trapping of IB could be used as an efficient method to inhibit constitutive NF-B activity, we initially employed W231.Bcl-XL B lymphoma cells that harbor constitutive NF-B (p50/c-Rel heterodimer) activity maintained by continuous degradation of IB (16, 19). This IB degradation is resistant to proteasome inhibitor treatment and independent of direct IKK-dependent phosphorylation of IB (described in ref. 16),2 two common anti-NF-B drug targets. As in other cell systems (13, 14), LMB treatment led to an increase in nuclear IB (Fig. 1A) that was bound to c-Rel (Fig. 1B). Nuclear trapping of these complexes consequently correlated with inhibition of constitutive p50/c-Rel activity (Fig. 1C). The IB-resistant p50/p50 homodimer complex remained resistant, consistent with the specificity of IB-dependent inhibition (20). These findings supported the notion that inhibition of nuclear export traps inactive, IB-sensitive NF-B in the nucleus. Ultimately, nuclear trapping led to the inhibition of continuous IB degradation (Fig. 1D and E) presumably by depleting the cytoplasmic pool of NF-B/IB complexes that are susceptible to the degradation pathway. Consistent with these studies, an IB protein with a mutant N-NES displayed slower kinetics of degradation than wild-type (WT) IB (Fig. 1F and G). These results imply that trapping IB in the nucleus retards its continuous cytoplasmic degradation and depletes constitutive NF-B activity.
Nuclear Trapping of IB Inhibits Constitutive NF-B Activity in Different Human Cancer Cells
Our observations in W231.Bcl-XL cells led us to hypothesize that nuclear export of IB can be targeted in multiple cancer cell types to generally inhibit constitutive NF-B activity. Initially, we analyzed the effects of LMB treatment on the activated B-cell-like, diffuse large B-cell lymphoma OCI-Ly3 cell line. It has been suggested that this cell line maintains constitutive NF-B activity through enhanced IKK activity that leads to the continuous degradation of IB via classic proteasome-dependent degradation (21). It has also been suggested that constitutive NF-B activity is required for the survival of this cell line (21). Similar to the W231.Bcl-XL cells, we wanted to determine whether inhibition of CRM-1-dependent nuclear export in the OCI-Ly3 cells could inhibit constitutive NF-B activity that is composed of multiple NF-B family members (Fig. 2A). Because of the complexity of the NF-B complexes present in these cells, supershift electrophoretic mobility shift assay (EMSA) analysis was employed to evaluate the effects of LMB treatment. These analyses revealed that p65-containing complexes, and to a lesser extent c-Rel-containing complexes, were inhibited by LMB treatment (Fig. 2B and D). In contrast, RelB-containing complexes were resistant to the effects of LMB (Fig. 2D, compare lanes 4, 8, and 12), consistent with the findings that these complexes are generally resistant to inhibition by IB (22). Subsequently, immunoprecipitation of p65 from nuclear extracts of LMB-treated OCI-Ly3 cells revealed that there was an increase in IB associated with p65 after treatment with LMB (Fig. 2C). In sum, these results in the OCI-Ly3 cells were consistent with the model that inhibition of IB nuclear export leads to an increase in nuclear NF-B/IB complexes and, consequently, a reduction in constitutive NF-B activity.
To assess whether inhibition of IB nuclear export could be used as a general strategy to inhibit constitutive NF-B activity in different cancer cell types, we next analyzed the inhibition of constitutive NF-B activity in another activated B-cell-like, diffuse large B-cell lymphoma (OCI-Ly10), breast cancer (MDA-MB-231), prostate cancer (DU145), and multiple myeloma (RPMI 8226) cell lines. Although the precise deregulation steps in signal transduction pathways are undefined in these cells, previous studies suggested that constitutive NF-B activity in the OCI-Ly10, MDA-MB-231, and DU145 cells results from an ongoing, classic IKK-dependent and proteasome-dependent IB degradation pathway (5, 7, 21). In contrast, constitutive NF-B activity in the RPMI 8226 cells seems to occur in a proteasome inhibitor resistant manner.2 Despite these different deregulated upstream mechanisms, EMSA and supershift analyses indicated that LMB treatment inhibited constitutive c-Rel or p65 NF-B complexes in all of these cell types (Fig. 3A-D). Similar to W231.Bcl-XL and OCI-Ly3 cells, inhibition of constitutive p65 activity was associated with increased levels of p65 associated with IB (Fig. 3E and F, compare lanes 1 and 2) and stabilization of IB protein, without any effect on the total levels of p65 (Fig. 3E and F). The similar findings in these disparate cancer cell types suggest that perturbation of CRM-1-dependent nuclear export of IB could be a general strategy to attenuate constitutive NF-B activation in different cancer types.
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FIGURE 3. LMB inhibits constitutive NF-B activity in multiple cancer cell types. A. Nuclear extracts from OCI-Ly10 cells were analyzed by supershift EMSA (left). OCI-Ly10 cells were left untreated, treated for 90 and 180 minutes with 20 ng/mL LMB, or treated for 180 minutes with 0.1% ethanol. Supershift EMSA of nuclear extracts was done using anti-p65 and c-Rel antibodies (right). Using the same quantitative analysis method as in Fig. 2B, the p65 supershifted complexes decreased by 50% and the c-Rel supershifted complexes decreased by 21% after treatment with LMB for 180 minutes. B. MDA-MB-231 cells were left untreated, treated with 20 ng/mL LMB for indicated times, or treated with 0.1% ethanol (ET) for 180 minutes. For the TNF- sample, cells were untreated or pretreated with 20 ng/mL LMB for 105 minutes followed by treatment with 10 ng/mL TNF- for 15 minutes. Nuclear extracts were analyzed by EMSA. Using the same analysis method as in Fig. 2B, the total NF-B activity decreased by 31% after treatment with LMB for 180 minutes. C. DU145 cells were examined similarly to the MDA-MB-231 cells in B. Total NF-B activity decreased by 24% after treatment with LMB for 180 minutes. D. Total extracts from RPMI 8226 cells were analyzed by supershift assays using indicated antibodies (left). RPMI 8226 cells were either left untreated or treated with 10 µmol/L benzyloxycarbonyl-leucyl-leucyl-leucinal (MG), 10 µmol/L clasto-lactacystin-ß-lactone (Lact), or 20 ng/mL LMB for 3 hours. For the TNF- samples, cells were left untreated or pretreated with the indicated inhibitors for 150 minutes followed by treatment with 10 ng/mL TNF- for 30 minutes. Total extracts were analyzed by EMSA. E. OCI-Ly10 cells were untreated or treated with 20 ng/mL LMB for 3 hours, and equivalent whole cell extracts were used for co-immunoprecipitation using anti-p65 or IB antibodies as described in Materials and Methods. F. MDA-MB-231 cells were analyzed as in E.
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Inhibition of Constitutive NF-B Activation via Inhibition of Nuclear Export Is Associated with the Induction of Apoptosis
Because we found that LMB prevents constitutive NF-B activation, which can provide an important survival function to cancer cells (1), we wanted to determine whether inhibition of constitutive NF-B activity by LMB could promote apoptosis of different cancer cell types. We examined the sensitivity of the OCI-Ly3 and RPMI 8226 cells to LMB treatment because they use different biochemical mechanisms to maintain continuous IB degradation (21).2 We found that treatment with LMB led to a significant increase in the percentage of cells undergoing apoptosis in both cell lines (Fig. 4A and B). A similar result was obtained with OCI-Ly10, DU145, and MDA-MB-231 cells albeit less effectively (data not shown).
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FIGURE 4. LMB induces apoptosis of cancer cell lines. A. OCI-Ly3 cells were left untreated, treated with 20 ng/mL LMB, or treated with 0.1% ethanol for 24 hours. Cells were fixed and stained with PI for cell cycle analysis as in Materials and Methods. Bars, SE. B. RPMI 8226 cells were treated and analyzed as in A. C. OCI-Ly3 cells were either left untreated or treated with 20 ng/mL LMB for 6 hours. RNA was prepared and reverse transcription-PCR was done as described in Materials and Methods. For cyclin D2 and FLIP, 50 ng RNA were used and amplified for 25 cycles, whereas IRF-4 required 25 ng RNA and an amplification of 25 cycles. These samples were quantified with Image J software to determine the average IPP of each band. Levels of gene expression were determined by comparing the average IPP of the indicated gene to the average IPP of GAPDH within a single reverse transcription-PCR reaction. Based on these analyses, the expression of cyclin D2, IRF-4, and FLIP were decreased by 16%, 34%, and 19%, respectively, in the presence of LMB.
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Previous studies by Davis et al. revealed that certain NF-B-regulated genes were up-regulated in the OCI-Ly3 cells (21). When we analyzed the levels of some of these NF-B-regulated genes in OCI-Ly3 cells exposed to LMB, we found evidence that the expression of cyclin D2, IRF-4, and FLIP were reduced (Fig. 4C). These findings together suggest that LMB-dependent inhibition of constitutive NF-B activity and its target gene expression is associated with apoptosis in certain cell lines.
LMB-Dependent Cell Death Requires Inhibition of NF-B Activity by IB
Because LMB can affect nuclear export of other proteins (23), not just IB, NF-B inhibition may only partially contribute to LMB-induced apoptosis in the above studies. To address this question, we examined the sensitivity of WT and p65–/– fibroblast cell lines to tumor necrosis factor- (TNF-)–inducible apoptosis in the absence or presence of LMB (Fig. 5A and B). Consistent with previous studies (24), p65–/–, but not WT, cells exhibited a marked increase in apoptosis on exposure to TNF- (Fig. 5A). Inhibition of NF-B activity by concurrent LMB treatment in the WT cells (Fig. 5C) led to a significant increase in apoptosis over TNF- treatment alone without any overt apoptotic induction by LMB treatment alone (Fig. 5A). There was a partial increase in the apoptotic response in p65–/– cells in the combined presence of TNF- and LMB (Fig. 5A), correlating with the inhibition of c-Rel-containing complexes in these samples (Fig. 5C, lane 7). These results suggest that the early onset of apoptosis can be induced by rapid LMB-dependent inhibition of NF-B activation before induction of a global cell death response to LMB treatment.
IB–/– fibroblast cells were then analyzed to determine whether LMB-mediated rapid cell death was governed through its specific effects on IB (Fig. 5A and B). It has been observed previously that inducible activation of NF-B in IB–/– cells can occur through IBß (12) and the level of IBß expression is higher in IB–/– MEF cells (Fig. 5B, lane 3) possibly to compensate for the deficiency in IB. Unlike IB, IBß does not contain a NES, undergo shuttling, or participate in postinduction repression, thereby making it refractory to LMB treatment (12). Consistent with this notion, LMB did not induce a marked apoptotic response in the presence of TNF- in IB–/– cells (Fig. 5A). As expected, LMB did not inhibit TNF--inducible NF-B activity in the IB–/– cells (Fig. 5C). Together, these results showed that LMB-dependent inhibition of NF-B activity could promote rapid apoptosis.
In summary, we provided several lines of evidence suggesting that inhibition of IB nuclear export could be a conserved target for inhibition of NF-B activity in multiple cancer cell types. Our studies suggest that LMB treatment can lead to the rapid onset of apoptosis in different cancer cell types arising from inhibition of nuclear export of IB and constitutive NF-B activity (Fig. 6). Notably, this acute induction of apoptosis via NF-B inhibition suggests that strategies combining minimal exposure to nuclear export inhibitors with other methods of chemotherapy may be effective at causing tumor regression while minimizing toxic side effects seen previously in clinical trials using LMB (25). Alternatively, it may be possible to develop other nuclear export inhibitors or those that specifically target the IB NES (e.g., by gene therapy strategies employing a N-NES mutant of IB) to improve therapeutic outcome. Overall, our findings provide strong support for inhibition of IB nuclear export as a viable approach to inhibit diverse NF-B cell survival pathways in a variety of cancer cells.
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Materials and Methods
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Cell Culture
Culture conditions for WEHI231 and W231.Bcl-XL cells have been described (16). OCI-Ly3 and Ly10 cells were provided by L. Staudt (NIH, Bethesda, MD) and maintained in the same conditions as WEHI231 cells. RPMI 8226 cells were maintained in DMEM, 10% fetal bovine serum, 10 mmol/L HEPES, and antibiotics as above and cultured in a 10% CO2 incubator. DU145, HEK293, and fibroblast cell lines were maintained in DMEM with 10% fetal bovine serum and antibiotics in a 10% CO2 incubator. MDA-MB-231 cells were maintained in RPMI with 10% fetal bovine serum and antibiotics in a 5% CO2 incubator.
Chemicals
LMB was provided by M. Yoshida (University of Tokyo, Tokyo, Japan). Cycloheximide was purchased from Sigma (St. Louis, MO). Benzyloxycarbonyl-leucyl-leucyl-leucinal was purchased from Peptide Institute, Inc. (Osaka, Japan). Clasto-lactacystin ß-lactone and human recombinant TNF- were purchased from Calbiochem (La Jolla, CA).
Antibodies
IgG antibodies to IB (C-21), actin (C-11), p65 (C-20), p50 (NLS), and RelB (C-19) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A monoclonal anti-HA.11 antibody was purchased from Covance (Berkeley, CA). The anti-Flag (M2) antibody was purchased from Scientific Imaging Systems (West Chester, PA). The -tubulin antibody was purchased from Oncogene Research (San Diego, CA). The c-Rel antibody was used as previously described (26). The p65 generated antibody was used as described previously (27). Horseradish peroxidase–conjugated protein A and horseradish peroxidase–conjugated anti-rabbit and anti-mouse antibodies were obtained from Amersham Pharmacia Biotech (Piscataway, NJ).
Mutagenesis and Infections
To generate a mutation of the IB N-NES (13), we did two-step PCR to substitute Ala residues for Met45, Val46, Leu49, and Ile52. The DNAs encoding both WT and mutant COOH-terminally hemagglutinin-tagged IB sequences were ligated into the pLHL-CA retroviral vector (28) and infection of W231.Bcl-XL cells was done as described previously (19).
Immunocytochemistry
Untreated and treated W231.Bcl-XL cells were resuspended in PBS-1% fetal bovine serum and spun on to glass slides for 8 minutes at 1,000 rpm in a Shandon Cytospin 2. Cells were then fixed in 3.7% formaldehyde at 4°C, washed, and permeabilized using PBS-0.2% Triton X-100. Following a blocking step in 2% goat serum, antibodies were added and left overnight. Staining was visualized with FITC-conjugated anti-rabbit and TRITC-conjugated anti-mouse antibodies. Cells were mounted with Prolong Antifade (Molecular Probes, Eugene, OR) and visualized and photographed using a Zeiss Axioplan epifluorescence microscope with the aid of fluorescein or rhodamine-specific filters.
Electrophoretic Mobility Shift Assay
EMSA was done as described (19) with either total cell or nuclear extracts (16). Oligonucleotides used for probes for the Ig intronic enhancer B site and the NF-Y site were purchased from Invitrogen (Carlsbad, CA) and double-stranded labeled probes were prepared as described (19). Probes for the Oct-1 site were purchased from Promega (Madison, WI) and end labeled as above. For supershift analyses, the indicated antibodies were added to the EMSA reactions and run normally.
Pulse-Chase Analysis
Pulse-chase experiments using W231.Bcl-XL cells and [35S]methionine-cysteine mixture (DuPont, Wilmington, DE) were done using an anti-IB antibody (C-21) as in ref. 29. Gels were dried for autoradiography and samples were quantified by PhosphorImager analysis using ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
Coimmunoprecipitation Assays
Coimmunoprecipitations using whole cell extracts were done as described previously (16). For coimmunoprecipitations using nuclear extracts, nuclear extracts were first prepared as described previously (16). The extract was diluted in TNEN buffer [10 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1 mmol/L EDTA, and 0.4% NP40] and split into fractions for immunoprecipitation.
Cell Cycle and Survival Analysis of Cell Lines
Cells were spun down at 1,600 rpm for 2 minutes, washed once in PBS, spun at 4,000 rpm for 5 minutes, fixed in 100 µL PBS with 900 µL 100% ethanol, and stored at –20°C overnight. Fixed cells were spun at 4,000 rpm for 5 minutes, washed once in 0.5 mL phosphate citric acid buffer (0.192 mol/L Na2HPO4, 4 mmol/L citric acid), spun at 4,000 rpm for 5 minutes, resuspended in 400 µL propidium iodide (PI) staining solution (1 mg/mL RNase A, 33 µg/mL PI, and 0.02% (v/v) NP40 in 0.1% PBS/bovine serum albumin/1 mmol/L EDTA), and stored at 4°C overnight. PI staining was analyzed by a FACScan or a FACSCalibur and the cell cycle profiles were examined using ModFit software.
To examine the survival of the fibroblast lines, treated cells were trypsinized and filtered, and PI (1 µg/mL) was added to each before analysis by a FACScan. The percentage of PI-positive cells was analyzed with CellQuest software.
Reverse Transcription-PCR Analysis
Cells (2.5 x 106) were treated for 6 hours, pelleted, washed once in PBS, and pelleted again. RNA was prepared immediately using Qiagen (Valencia, CA) RNeasy Mini Kit and Qiashredder according to manufacturer's instructions. Primers were chosen from the genes identified in OCI-Ly3 diffuse large B-cell lymphoma by Dr. L. Staudt (30). Reverse transcription-PCR reactions were then done with either 25 or 50 ng total RNA using the Qiagen One-Step reverse transcription-PCR kit according to the manufacturer's instructions. The following primers were used for each gene: cyclin D2 (5'-GTCTGTGAGGAACAGAAGTGC-3' and 5'-CGGTGGCACACAGAGCAATG-3', 376 bp), IRF-4 (5'-GTTGCCAGGTGACAGGAACC-3' and 5'-CTGACAAGAACTGCTGTGTG-3', 462 bp), FLIP (5'-GTGCTGATGGCAGAGATTGG-3' and 5'-GTTGAGCGCCAAGCTGTTCC-3', 379 bp), and GAPDH (5'-GTCTTACTCCTTGGAGGCCATG-3' and 5'-ACCCCTTCATTGACCTCAACTAC-3', 
750 bp). Samples were run on agarose gels, stained with ethidium bromide, and quantified using Image J software (NIH).
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Acknowledgements
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We thank Drs. E. Davis and L. Staudt for the OCI-Ly3 and OCI-Ly10 cell lines, Dr. D. Baltimore (Cal Tech) for the p65–/– and IB–/– cells, Dr. M. Yoshida for his gift of LMB, and Dr. C. Berchtold (University of Wisconsin-Madison) for statistical analyses.
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Notes
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1 NIH grants R01CA-81065 and R01-CA77474 (S. Miyamoto), American Heart Association predoctoral fellowship (S. O'Connor), and NIH predoctoral training grant T32GM07215 (S. Shumway and S. O'Connor). 
2 O'Connor S and Miyamoto S. Evidence for a phosphorylation-independent role for Ser 32 and 36 of IB in proteasome inhibitor resistant (PIR) degradation in B cells, submitted for publication.
Received August 25, 2004;
revised November 5, 2004;
accepted December 10, 2004.
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Abstract
Introduction
